Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Two different modes of inhibition of the rat brain Na+,K+-ATPase by triterpene glycosides,psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na⁺,K⁺-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na⁺,K⁺-ATPase with I₅₀ values of 1×10⁻⁴ M and 3×10⁻⁴ M, respectively. PsA significantly stimulated [³H]ATP binding to Na⁺,K⁺-ATPase, weakly increased [³H]ouabain binding to the enzyme, and inhibited K⁺-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [³H]ATP binding to Na⁺,K⁺-ATPase, and had no effect on [³H]ouabain binding to the enzyme. K⁺-Phosphatase activity was inhibited by PsB in parallel with Na⁺,K⁺-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na⁺,K⁺-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

The Role of Na+/K+-ATPase during Chick Skeletal Myogenesis

The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na⁺/K⁺-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na⁺/K⁺-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM) and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na⁺/K⁺-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na⁺/K⁺-ATPase.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney

The rates of association of [³H]ouabain to Na⁺,K⁺-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na⁺,K⁺-ATPase being the fastest. Although the major determinant of the affinity of the Na⁺,K⁺-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na⁺,K⁺-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I₅₀ (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na⁺,K⁺-ATPase activity, approached the value calculated from [³H]ouabain binding. The ratio of the I₅₀ value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na⁺,K⁺-ATPase is not the reason for the faster dissociation rate of this enzyme.     

Overexpression of Na+/K+-ATPase Parallels the Increase in Sodium Transport and Potassium Recycling in an In Vitro Model of Proximal Tubule Cellular Ageing

Na⁺/K⁺-ATPase plays a key role in the transport of Na⁺ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na⁺ and K⁺ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na⁺/K⁺-ATPase activity and K⁺ conductance were evaluated using electrophysiological methods. Na⁺K⁺-ATPase α₁– and β₁-subunit expression was quantified by Western blot techniques. Na⁺/H⁺ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na⁺ transport, which results from an increase in the number of Na⁺/K⁺-ATPase α₁- and β₁-subunits, in the membrane. Increases in Na⁺/K⁺-ATPase activity were accompanied by increases in K⁺ conductance as a result of functional coupling between Na⁺/K⁺-ATPase and basolateral K⁺ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na⁺/H⁺ exchanger activity were observed. H₂O₂ production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H₂O₂ had no effect on Na⁺/K⁺-ATPase expression. Ouabain (100 nM) increased α₁-subunit, but not β₁-subunit, Na⁺/K⁺-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Tryptic inactivation of the ouabain binding site of canine kidney Na+,K+-ATPase and its effect on catalytic function.

Trypsin treatment of the purified Na⁺, K⁺-ATPase from canine renal outer medulla causes loss of ADP-ATP exchange activity when digestion takes place in 0.1 M KCl. Activity surviving this treatment remains inhibitable by ouabain. Addition of ATP to such digestion mixtures stabilizes the Na⁺, K⁺-ATPase in a different conformation (Na⁺-form). Under these conditions ADP-ATP exchange activity is protected, and becomes ouabain-insensitive. Quantitative analysis of the cleavage products and rates of loss of ouabain binding and exchange activity suggest that catalytically inactive trypsinolysis products can bind ouabain, and that the 85,000 dalton fragment associated with ouabain-insensitive ADP-ATP exchange activity cannot bind ouabain. Cleavage to produce the 85,000 dalton fragment therefore destroys the ouabain binding site.     

Some in vivo and in vitro effects of ethacrynic acid on renal Na+,K+ -ATPase

Binding of [¹⁴C]ethaerynic acid [EA]at concentrations of EA from 10^?4m to 10^?2m to a membrane preparation containing Na⁺,K⁺-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na⁺ or K⁺, but was not affected by Mg²⁺ and/or ATP. [¹⁴C]EA significantly bound to a microsomal preparation with low Na⁺,K⁺-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na⁺. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [¹⁴C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [³H]ouabain binding to medullary and cortical Na⁺,K⁺-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [¹⁴C]EA binding to Na⁺,K⁺-ATPase was not affected by pretreatment of the preparation with ouabain (10^?6m to 5 × 10^?4m). EA reduced the sensitivity of [³H]ouabain binding to the enzyme preparation to Na⁴ and K⁺.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na⁺,K⁺-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [³H]ouabain to Na⁺,K⁺-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10^?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na⁺,K⁺-ATPase is a pharmacological receptor for ethacrynic acid.     

Characterization of Na+, K+-ATPase in Cultured and Separated Neuronal and Glial Cells from Rat Cerebellum

Abstract: The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na ⁺, K⁺- ATPase; EC 3.6.1.3) were examined in cultures and peri- karya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synapto- somal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na ⁺, K⁺-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3–15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the a'strocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na ⁺, K⁺-ATPase (apparent A_j values of about 10–⁷M and 10–⁴-10–⁵M, respectively), the high- affinity form accounting for 60–75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na ⁺,K ⁺ - ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na⁺,K⁺-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na ⁺, K⁺-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into acc.ount the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na⁺, K⁺-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of ⁸⁶Rb into “live cells” was estimated as a measure of in situ ion pump activity. Besides the interaction with ouabain, the K⁺ dependence of the Na⁺, K⁺-ATPase activity was also investigated in crude particulate preparations from cultured cerebellar neurones and astrocytes. Differences were observed as nearly maximal enzyme activity was obtained in the as- trocyte preparations at 1 mM KCl, when only about one- third of the maximal activity was displayed by the cultured nerve cells.     

Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na⁺ overload was found to accompany the cell death. Depletion of Na⁺ in culture medium or pretreatment of cells with the Na⁺/K⁺-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na⁺/K⁺-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na⁺/K⁺-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na⁺/K⁺-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.     

Extracellular Allosteric Na Binding to the Na,K-ATPase in Cardiac Myocytes

Whole-cell patch-clamp measurements of the current, I_p, produced by the Na⁺,K⁺-ATPase across the plasma membrane of rabbit cardiac myocytes show an increase in I_p over the extracellular Na⁺ concentration range 0–50 mM. This is not predicted by the classical Albers-Post scheme of the Na⁺,K⁺-ATPase mechanism, where extracellular Na⁺ should act as a competitive inhibitor of extracellular K⁺ binding, which is necessary for the stimulation of enzyme dephosphorylation and the pumping of K⁺ ions into the cytoplasm. The increase in I_p is consistent with Na⁺ binding to an extracellular allosteric site, independent of the ion transport sites, and an increase in turnover via an acceleration of the rate-determining release of K⁺ to the cytoplasm, E2(K⁺)₂ → E1 + 2K⁺. At normal physiological concentrations of extracellular Na⁺ of 140 mM, it is to be expected that binding of Na⁺ to the allosteric site would be nearly saturated. Its purpose would seem to be simply to optimize the enzyme’s ion pumping rate under its normal physiological conditions. Based on published crystal structures, a possible location of the allosteric site is within a cleft between the α- and β-subunits of the enzyme.     

Crosstalk between Na+,K+-ATPase and a volume-regulated anion channel in membrane microdomains of human cancer cells

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na⁺,K⁺-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na⁺,K⁺-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na⁺,K⁺-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na⁺,K⁺-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.     

Effect of cold acclimation on Na+/K+ transport in hamster liver cells

Summary The effects of prolonged cold exposure of Syrian hamsters on liver membrane (Na⁺/K⁺-ATPase activity and on liver intracellular K⁺ levels was examined. Membrane preparations from cold-acclimated hamsters (6°C for 3 weeks) exhibited significantly higherV _max values for (Na⁺/K⁺)-ATPase and significantly greater ouabain binding. These data support the view that in the liver of these cold-exposed hamsters, there is an increase in the number of operational pumps. The fact that the intact liver cells (isolated via liver perfusion) from the cold-acclimated hamsters: (a) did not have higher concentrations of intracellular K⁺ (despite the presence of more operational pumps); and (b) exhibited greater rates of K⁺ loss when the pumps were inhibited by maximal ouabain suggests that the K⁺ leak across the liver cell plasma membrane is increased in the cold-acclimated hamsters. Although the physiological significance of these results needs further evaluation, these membrane changes may be of adaptive value for hibernation.Abbreviations CA cold-acclimated - P _i inorganic phosphate - KRB Krebs-Ringer-bicarbonate buffer - BSA bovine serum albumin - ECF extracellular fluid - ICF intracellular fluid - dcs dry cell solid - N nitrogen     

Age-dependent effect of ouabain on renal Na+,K+-ATPase

AimsThis study examines the effect of chronic ouabain-treatment on renal Na⁺ handling in 12-week and 52-week old rats.Main methodsWistar Kyoto rats aged 5 weeks or 45 weeks were treated with ouabain or vehicle during 7 weeks. Blood pressure was measured in conscious animals throughout the study. After 7 weeks of treatment urinary electrolyte concentration, Na⁺,K⁺-ATPase activity and α₁-subunit expression were determined in 12-week and 52-week old rats.Key findingsIn 12-week and 52-week old rats ouabain produced a significant increase in systolic blood pressure. Although no differences were observed in Na⁺ excretion in these animals, 12-week old ouabain-treated rats had lower Na⁺,K⁺-ATPase activity in proximal tubules. However, 12-week old ouabain-treated rats had decreased fractional excretion of Na⁺. In proximal tubules of 52-week old rats Na⁺,K⁺-ATPase activity did not differ between vehicle and ouabain-treated groups.SignificanceOur results show that in Wistar Kyoto rats renal response to ouabain treatment may be age-dependent and that the hypertensive effect of ouabain is independent of the effect on renal Na⁺,K⁺-ATPase.